Relationship Between Expressive Of UHRF1and Radiosensitivity In Esophageal Squamous Cell Carcinoma

Esophageal carcinoma is one of the most common malignantdiseases in the world, with high incidence, poor cure rate and highmortality characteristics.90％of esophageal tumors are esophagealsquamous cell carcinoma (ESCC) in China, even in Asia-pacific region.Currently radiotherapy is the mainstay in the treatment of esophagealcancer, but the local failure has remained a major concern, with persistentor recurrent disease being reported in around60-80%of patients, and anoverall5-year survival rate of10％. It is still a challenged question howto enhance radiosensitivity of esophageal carcinoma cells, to decreaselocal recurrent rate, and to increase local control rate and long-timesurvival.With the development of cellular and molecular biology, So far,several genes and their products have been found out which areresponsible for radiosensitivity, including cell cycle gene, apoptosis gene,DNA damage repair gene and et al, indicating that the chang of genepattern can be a big factor affecting radiosensitivity. Ubiquitin-like withPHD and ring finger domains1(UHRF1) is a novel nucleoprotein genewhich associated with cell growth. It plays important roles in manybiological processes including proliferation, cycle regulation andradiosensitivity. UHRF1protein is a multi-domained protein which canregulate gene expression through epigenetic mechanisms including DNAmethylation, histone deacetylation, histone methylation and possiblyhistone ubiquitination. Recently, UHRF1has become one of the majortargets of cancer researches. Clinical researchs have been showed thatUHRF1is overexptessed in many tumor tissues and associated withmalignant biological behavior and poor prognosis. It has been found that UHRF1plays important roles in radioresistance. UHRF1-null mouse EScells are more sensitive to X-ray, UV light andN-methyl-N″-nitro-N-nitrosoguanidine (MNNG) than wild-type orUHRF1+/cells. Enhanced expression of UHRF1reducesradiosensitivity of breast cancer MDA-MB-231cells and cervical cancerHeLa cells to ionising radiation. It is suggested that UHRF1can be anovel target to increase the radiosensitivity. Neverthless, the roles ofUHRF1in esophageal squamous cell carcinoma (ESCC) has not beenwell illustrated. RNA interference (RNAi) is a quick, simple andeconomical method for gene inhibition, and has brought new hope forgene treatments for tumor. Lentivirus vector has its unique advantages tointegrate its genes into the human host’s-ensuring the virus’s permanence.In present study, the expression of UHRF1in esophageal carcinomatisssue and normal mucous membrane of esophagus tissue was observedrespectively，and the relationship between UHRF1expression levels withthe clinicopathologic characteristic and radiotherapy effect of esophagealcarcinoma were further analysized. Secondly, lentivirus-mediatedrecombinant plasmid shRNA targeting UHRF1was constructed andtransfected into esophageal carcinoma cell line TE-1to inhibit theexpression of UHRF1, and its effects on biological behiviors andradiosensitivity of esophageal carcinoma were investigated.Part1The relationship between UHRF1expression and radiotherapyeffect in ESCCObjective: To explore the relationship between UHRF1expressionand radiotherapy effect in ESCCMethods: Surgical specimens and matched noncancerous tissuesfrom50ESCC patients and three kinds of cell lines TE-1, TE-13, Eca109were examined for UHRF1expression by Reversetranscription-polymerase chainreaction (RT-PCR) and Western blotanalysis. In addition,61biopsy specimens of ESCC patients treated withdefinitive radiotherapy were also detected for UHRF1expression by immunohistochemistry (IHC), and the correlation of UHRF1expressionwith the patient’s radiotherapy effect and clinicopathologic characteristicwere analyzed.Results: The RT-PCR, Western blot and IHC results demonstratedthat the expression level of UHRF1was significantly higher in ESCCtissues and cells than in matched noncancerous tissues. Both UHRF1mRNA and protein expression in the ESCC tissues were significantlycorrelated with invasion depth, lymph node metastasis and histologicalgrade. However, there was no significant difference in UHRF1expressionbased on age, gender, tumor length and location. In the RT group, theresponse of tumors with a high level expression of UHRF1wassignificantly poorer than that of tumors with low expression.Conclusion: UHRF1is overexpressed in ESCC tissues and cells,and closely related with the development, migration and radioresistanceof ESCC.Part2Construction of lentivirus-mediated recombinant plasmidshRNA targeting UHRF1and its effects on biological behiviors ofesophageal carcinoma cellsObjective: To construct the lentivirus-mediated recombinantplasmid shRNA targeting UHRF1and analylize its effects on biologicalbehiviors of esophageal carcinoma cellsMethods: A totle of4pairs of antisense oligonucleotide fragmentswere designed and annealed, and then introduced into RNA interferingexpression vector pGCSIL, and the recombinant expression vectorpGCSIL-GFP-UHRF1-shRNA was successfully constructed. Then,lentiviral packaging vectors and pGCSIL-GFP-UHRF1-shRNA were thentransfected into tool cells, and the lentiviral supernatants containingpGCSIL-GFP-UHRF1-shRNA were harvested later after transfection.Then, TE-1cells were transfected with lentiviral supernatants, andstablely expressing pGCSIL-GFP-UHRF1-shRNA was screened withfluorescence microscope. The mRNA and protein expression of the UHRF1gene in TE-1cells were investigated after transfection byRT-PCR and Western blot, respectively. Finally, the proliferation, cycledistribution, apoptosis and invasion ability of TE-1cells were detectedwith MTT, flow cytometry, Transwell chamber extraneous migratoryexperiment after transfection.Results: A totle of4siRNA expression vectorpGCSIL-GFP-UHRF1-shRNA1, pGCSIL-GFP-UHRF1-shRNA2,pGCSIL-GFP-UHRF1-shRNA3and pGCSIL-GFP-UHRF1-shRNA4were successfully constructed using gene recombination technology, inwhich pGCSIL-GFP-UHRF1-shRNA1was screened as most inhibitoryeffect. Expression levels of the UHRF1gene in TE-1cells transfectedwith pGCSIL-GFP-UHRF1-shRNA1was significantly lower than thosein control and untransfected groups. After transfection the proliferationand migration of TE-1cells were inhibited, and cell cycle arrested inG0/G1phase. In addition, the apoptotic rate of TE-1cells significantlyincreased.Conclusion: Lentivirus-mediated shRNA targeting UHRF1cansilence the expression of UHRF1, attenuate the proliferation and invasion,change the distribution of cycle and induce the apoptosis in TE-1cells.Part3The effect of lentivirus-mediated shRNA targeting UHRF1onradiosensitivity of esophageal carcinoma cellsObjective: To investigate the effect of lentivirus-mediated shRNAtargeting UHRF1on radiosensitivity of TE-1cells and its mechanisms.Methods: After transfection with lentivirus-mediated shRNAtargeting UHRF1, TE-1cells were treated with X-ray irradiation. Cloneformation assay was used to detect the radiosensitivity of TE-1cells aftertransfection. Cell cycle distribution, apoptosis and cell cycle-relatedprotein-CyclinB1, apoptosis-related protein-Bax, Caspase-8, DNAdamage repair protein-Ku70, Ku80, DNA-PKcs, RAD51were detectedby flow cytometry and Western blot to analyze radiosensitivitymechanism. Results: Clone formation assay showed that the survival fractionand radiobiology parameters (D0, Dq, N and SF2) values inUHRF1-shRNA transfected group were much lower than those innon-transfection and mock-vehicle groups after0-8Gy irradiation. Thesensitization enhancing ratio in UHRF1-shRNA transfected group were1.53(D0ratio) and1.95(Dq ratio). Further analysis showed that G2/Marrest and apoptosis were induced by8Gy irradiation. Meanwhile, cellcycle-related protein-CyclinB1expression was down-regulated;apoptosis-related protein-Bax, Caspase-8expression and DNA damagerepair protein-Ku70, Ku80, DNA-PKcs, Rad51expression wereupregulated by irradiation. After UHRF1-shRNA1transfection,irradiation-induced G2/M arrest was decreased; irradiation-inducedapoptosis was increased. Besides that, endogenous andirradiation-induced Bax and Caspase-8expression were upregulated, andendogenous and irradiation-induced Ku70and Ku80expression weredownregulated. But the expression of CyclinB1, DNA-Pkcs and Rad51were not changed.Conclusion: UHRF1-shRNA1transfection can enhance theradiosensitivity of TE-1cells. Its mechanism is correlated with regulatingcell cycle-ralated protein, apoptosis ralated protein, DNA damage repairprotein expression to decrease G2/M arrest and DNA damage repaircapacity, and increase apoptosis.